Presence Of Nitro Blue Tetrazolium Research Articles

Efficient and green oxidation of 2-aminophenol catalyzed by tetra-(p-methoxyphenyl) porphyrin complexes anchored on chitosan in bicarbonate solution

The study explored the catalytic activities of chitosan-supported tetra (p-methoxyphenyl) porphyrin complexes 1–3 in the heterogeneous activation of the aerobic oxidation dimerization of 2-aminophenol (OAP) to 2-aminophenoxazine-3-one (APX) in the presence of bicarbonate, simulating the function of phenoxazinone enzyme synthase. The oxidation reaction was followed by recording the UV–vis spectra of the reaction mixture with time at λmax 433 nm. All chitosan-supported metalloporphyrin complexes 1–3 exhibited effective catalytic activities for OAP oxidation. Under optimal conditions, the chitosan-supported Cu(II) (Tp-OCH3PP) complex displayed the highest catalytic efficiency. Various parameters influencing the catalytic activity of Cu(II) (Tp-OCH3PP)/CTS 1 were studied. The observed rate constant of OAP oxidation exhibited a direct correlation with the concentration of supported catalyst 1 and followed Michaelis–Menten kinetics, indicating saturation of catalyst sites with increasing OAP concentration. The study investigated the impact of temperature, bicarbonate concentration, dissolved oxygen, and the reaction mechanism. Oxidation reaction of OAP catalyzed by 1 in the presence of nitro blue tetrazolium (NTB) revealed no superoxide anion O2−• was formed as a reactive species during the reaction. The chitosan-supported Cu(II) (Tp-OCH3PP) complex shows high catalytic stability and no significant changes up to the fifth run.

DECREASE IN THE CONTENT OF REACTIVE OXYGEN SPECIES DURING INACTIVATION OF THE RESPIRATORY COMPLEX I IN ARABIDOPSIS CELLS

Background. The mitochondrial electron transport chain, in particular the respiratory complex I, is one of the main sources of reactive oxygen species (ROS) in living cells. Suppression of the complex I activity in human cells through chemical inhibition or mutations that disrupt the functionality of this complex leads to a significant increase in the content of ROS. The complex I mutants are also known for plants; however, it is still not clear whether the suppression of the activity of this complex leads to an increase in the level of ROS and the development of oxidative stress.
 Purpose. To study the level of superoxide, hydrogen peroxide and sensitivity to prooxidants in Arabidopsis ndufs4 cells with complex I inactivated by insertional mutagenesis.
 Materials and methods. A suspension culture of wild-type Arabidopsis cells and a line with knockout the NDUFS4 complex I subunit was used. The level of hydrogen peroxide in the cells was determined using dichlorofluorescein. The superoxide content was estimated by cell staining in the presence of nitroblue tetrazolium.
 Results. It has been shown that inactivation of the NADH-dehydrogenase complex in Arabidopsis cells due to the absence of one of its subunits leads to a decrease in the content of superoxide and hydrogen peroxide. It was also shown that the level of ROS in cells treated with the menadione and hydrogen peroxide increased several times in wild-type cells, but remained almost unchanged in ndufs4 cells.
 Conclusion. Suppression of the activity of the NADH dehydrogenase complex in plant cells, but not in animal cells, leads to a decrease in the content of both hydrogen peroxide and superoxide. Cells of ndufs4 line have an increased ability to detoxify ROS, possibly associated with the constant mobilization of antioxidant defense systems.

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

The chloroplast outer envelope protein OEP37 is a member of the growing beta-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance (lambda) of Lambda= 500 picosiemens (symmetrical 250 mm KCl). The OEP37 channel is cation-selective (P(K+)/P(K-) = 14:1) with a voltage-dependent open probability maximal at V(mem) = 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d = 3.0 nm and the restriction zone by d = 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.

New food antioxidant additive based on hydrolysis products of lactose

The hydrolysis products of lactose, D-glucose and D-galactose, were oxidized in two consecutive steps using appropriate bimetallic catalysts (Bi–Pd, Bi–Pt on charcoal) to the corresponding 2-ketoaldonic acids. The latter were further processed to provide two stereoisomers of L-ascorbic acid (vitamin C), D-ascorbic acid (D-lyxoascorbic acid) and erythorbic acid (D-araboascorbic acid). The antioxidant activity of these two isomers as well as that of the equimolar mixture thereof was evaluated by measuring their reducing activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) and their scavenging activity of O2− species in the presence of NBT (Nitro Blue Tetrazolium). The compounds and their equimolar mixture were found to exhibit the same antioxidant activity as L-ascorbic acid suggesting that they may be used as cheap alternatives to L-ascorbic acid as food antioxidant additives when the vitamin C activity is not required.

The photochemistry of dipyridamole

The photochemical reactions of dipyridamole (DIP), a healing agent, was studied to predict the photobiological implications of its use which will be the subject of a future article. Its photolysis product under UV-A and aerobic conditions was isolated and identified. DIP was shown to be photostable under inert atmosphere (argon). On the other hand, under the same conditions a process of electron transfer was detected in the presence of nitro blue tetrazolium (NBT) which can be inhibited by oxygen. The photodegradation of DIP occurs probably via a type II mechanism involving irreversible trapping of self-photogenerated reactive oxygen species (ROS). This fact could be indicative of its antioxidant activity. The formation of singlet oxygen and superoxide during the DIP photodegradation makes one suspicious of a possibility that DIP could also be involved in oxidative stress in biological systems. Further studies on biological systems will contribute to elucidate this probable dual behavior.

Potential role of a membrane-bound NADH oxidoreductase in nitric oxide release and arterial relaxation to nitroprusside.

The site of metabolism in vascular smooth muscle responsible for the release of nitric oxide (NO) from nitroprusside is not well established. In this study we observed that a membrane-bound NADH oxidoreductase in the pulmonary artery activates nitroprusside to release NO, and we examined whether this process could potentially participate in relaxation to nitroprusside. Relaxation to nitroprusside in bovine calf pulmonary artery is inhibited by a scavenger of NO and by an antagonist of NO stimulation of guanylate cyclase. A flavoprotein probe that inhibits pulmonary artery NADH oxidoreductase (1 micromol/L diphenyliodonium) and electron acceptors for NADH oxidoreductase (0.3 mmol/L nitroblue tetrazolium and 0.1 mmol/L ferricyanide) inhibited pulmonary artery relaxation to nitroprusside, but not to nitroglycerin. Pulmonary arteries were observed to promote the release of NO from nitroprusside in vitro, and NO release was inhibited by the presence of nitroblue tetrazolium, ferricyanide, and diphenyliodonium. In homogenates of pulmonary arteries, NADH (0.1 mmol/L) increased the release of NO from nitroprusside by approximately 6-fold, whereas NADPH, mitochondrial substrates, and other redox cofactors had minimal effects on NO release, and the action of NADH on nitroprusside was inhibited by nitroblue tetrazolium, ferricyanide, and diphenyliodonium. A membrane fraction enriched in NADH oxidoreductase activity showed a NADH-dependent release of NO from nitroprusside; nitroprusside caused NADH consumption, and it also inhibited the NADH-dependent reduction of nitroblue tetrazolium. Thus, a membrane-bound NADH oxidoreductase appears to contribute to the release of NO from nitroprusside, but not nitroglycerin, in calf pulmonary artery.

Electron Transfer Processes in the Reactivity of Nonsteroidal Anti‐inflammatory Drugs in the Ground and Excited States

The nonsteroidal anti-inflammatory drugs (NSAID), naproxen, sulindac and indomethacin, were shown to donate electrons to nitro blue tetrazolium (NBT) when irradiated with UV light in deoxygenated aqueous buffer solution (pH 7.4, 30 degrees C). The reaction was monitored spectrophotometrically by the appearance of the diformazan reduction product from NBT. The electron transfer process facilitates the decomposition of the drugs. Naproxen in the presence of NBT is photodegraded principally to the alcohol (2-[1-hydroxyethyl]-6-methoxynaphthalene) at a rate approximately 20-fold faster than when irradiated alone in deoxygenated conditions. The photoproduct from naproxen also participates in the electron transfer to NBT but at a much slower rate than naproxen. Irradiation of sulindac or indomethacin in the presence of NBT caused the slow photoreduction of NBT to diformazan. In the absence of NBT, indomethacin and sulindac are essentially unreactive when irradiated in aqueous solution. The ability of a number of NSAID to act as electron donors in their ground state was studied by observing their oxidation by potassium peroxodisulfate in pH 7.0 phosphate buffer at 50 degrees C. The HPLC analysis of the drug remaining showed that the 2-arylpropionic acid NSAID (naproxen, ibuprofen, ketoprofen and suprofen) reacted at a rate equivalent to the thermal decomposition of peroxodisulfate. The major products were the same as detected in the photooxidation of these drugs, resulting from decarboxylation and oxygen addition but also included a dimeric compound. On the other hand, the NSAID that do not contain the propionic acid substituent all reacted more slowly with peroxodisulfate, enabling specific reaction rate constants to be evaluated.

Electron Transfer Processes in the Reactivity of Nonsteroidal Anti-inflammatory Drugs in the Ground and Excited States

The nonsteroidal anti-inflammatory drugs (NSAID), naproxen, sulindac and indomethacin, were shown to donate electrons to nitro blue tetrazolium (NBT) when irradiated with UV light in deoxygenated aqueous buffer solution (pH 7.4, 30°C). The reaction was monitored spec-trophotometrically by the appearance of the diformazan reduction product from NBT. The electron transfer process facilitates the decomposition of the drugs. Naproxen in the presence of NBT is photodegraded principally to the alcohol (2-[1-hydroxyethyl]-6-methoxynaphthalene) at a rate approximately 20-fold faster than when irradiated alone in deoxygenated conditions. The photoproduct from naproxen also participates in the electron transfer to NBT but at a much slower rate than naproxen. Irradiation of sulindac or indomethacin in the presence of NBT caused the slow photoreduction of NBT to diformazan. In the absence of NBT, indomethacin and sulindac are essentially unreactive when irradiated in aqueous solution. The ability of a number of NSAID to act as electron donors in their ground state was studied by observing their oxidation by potassium peroxodisulfate in pH 7.0 phosphate buffer at 50°C. The HPLC analysis of the drug remaining showed that the 2-arylpropionic acid NSAID (naproxen, ibuprofen, ketoprofen and suprofen) reacted at a rate equivalent to the thermal decomposition of peroxodisulfate. The major products were the same as detected in the photooxidation of these drugs, resulting from decarboxylation and oxygen addition but also included a dimeric compound. On the other hand, the NSAID that do not contain the propionic acid substituent all reacted more slowly with peroxodisulfate, enabling specific reaction rate constants to be evaluated.

Evidence for azidyl radical initiated olefin isomerization. One-way isomerization of (Z)-urocanic acid

(Z)-Urocanic acid (cis-UA), unlike its (E) isomer (trans-UA), undergoes photoisomerization in the presence of nitro blue tetrazolium (NBT 2+) and sodium azide. Evidence is presented that the photoisomerization involves the reversible addition of the azidyl radical to the double bond.

Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH 10.35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a half-life of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.

Characterization of the Glycine-Dependent Redox-Cycling Activity in Animal Fluids and Tissues Using Specific Inhibitors and Activators: Evidence for Presence of PQQ

Pyrroloquinoline quinone, a redox cofactor first isolated from bacteria, efficiently catalyzes the nonenzymatic oxidation of glycine in the presence of nitroblue tetrazolium. We report that certain metalic cations and heterocyclic aromatic cations, like the N-methyl phenazonium cation and aryl-iodonium compounds, strongly and specifically inhibit this redox-cycling activity. The inhibition by metal cations is reversed by Tiron and that of the aromatic cations by Tiron and thyroxine. These inhibitors and activators affect authentic PQQ and the redox-activity of putative PQQ isolated from biological sources in a similar manner. This indicates that pyrroloquinoline quinone occurs naturally in animal tissues and fluids.

Development of an automatic machine for in Situ hybridization and immunohistochemistry

An instrument for the automation of in situ hybridization and immunohistochemistry has been developed. This machine is capable of analyzing 20 microscope glass slides via all of the steps required for colorimetric in situ hybridization or immunohistochemistry. The slides are placed specimen-side down on a specialized Teflon slide-holder set in the reaction chamber of the machine. The system uses a unique type of capillary action between the slide and the holder. The holder has two small holes and is designed to apply, incubate and sequentially add and remove reagents from the slide surface. The system performs the complete processes of in situ hybridization and immunohistochemistry from dewaxing to colorization. Some applications were carried out using this instrument. Cultured cells infected with cytomegalovirus, adenovirus, or herpes simplex virus were hybridized with homologous biotinylated probes, and showed strong purple signals with alkaline phosphatase in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Automatic in situ hybridization using other colorimetric detection systems (e.g., peroxidase-labeled probes/diaminobenzidine/H 2O 2) was also examined in cells infected with Chlamydia trachomatis and in paraffinembedded hepatic tissue sections from patients with hepatitis. For conventional immunohistochemical staining, formalin-fixed and paraffin-embedded tissues were used. Glial fibrillary acidic protein and γ-immunoglobulins were detected automatically in human brain white matter and tonsillar tissues, respectively, as peroxidase-based reddish signals. The intensity of staining was equal to that achieved by manual methods.

A cytochemical staining procedure for succinate dehydrogenase activity in pre-ovulatory mouse oocytes embedded in low gelling temperature agarose.

We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells.

Alkaline phosphatase activity in the brain of the American cockroachPeriplaneta americana L.

The supra- and suboesophageal ganglia of the American cockroach contain material which catalyses the alkaline hydrolysis (pH 9.5) of 5-bromo-4-chloro-3-indolyl phosphate in the presence of Nitro blue tetrazolium. Histochemical studies on unfixed cryostat sections indicate that this type of alkaline phosphatase is restricted to discrete regions in the cockroach brain. Highest enzyme activity is encountered in the mushroom bodies, central body, antennal glomeruli and specific parts of some distinct neural connections including the optic nerve, antennal nerve, circumoesophageal connectives and nerves leaving the suboesophageal ganglion. Tissue fixation by use of formaldehyde-type fixatives, as well as routine paraffin-embedding, completely destroy all histochemically detectable enzyme activity. Native polyacrylamide gradient electrophoresis suggests that the alkaline phosphatase activity is present as multiple isozymic forms, which show up in the 120-130 kD range of standard proteins. Enzyme activity becomes undetectable after fixation (trichloroacetic acid, formaldehyde containing fixatives) of electrophoretically separated native proteins, as well as after electrophoresis in denaturing conditions (SDS and beta-mercapto-ethanol, boiling). However, the enzyme activity remains virtually unaffected after storage of the sample for prolonged periods at -20 to -80 degrees C.

Preparation of antibodies against xanthine oxidase from human milk

1. 1. Human xanthine oxidase [XO; EC 1.2.3.2] was isolated by a non-proteolytic method from fresh human milk. Final purification of the protein was achieved by hydroxyapatite chromatography. Most (<95%) of the enzyme was released in the 0.40 M phosphate fraction at pH6.8. 2. 2. The specific activity of this preparation was found to be 0.047 μM min −1mg −1 with xanthine as substrate. 3. 3. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two subunits, each with a mol. wt ~122 kDa. 4. 4. On non-denaturing acrylamide gels both of these subunits exhibited oxidase-like activity with xanthine as substrate in the presence of nitroblue tetrazolium and molecular oxygen. 5. 5. Immunoconjugates of XO were prepared by the keyhole limpet hemocyanin (KLH)- and glutaraldehyde-crosslinking techniques. 6. 6. Polyclonal antibodies to XO were raised by i.m. injection of these conjugates into female New Zealand rabbits. 7. 7. Western blot analysis using the semi-dry technique was employed to confirm the specificity of the antibody.

Iron autoxidation in Mops and Hepes buffers.

Iron autoxidation in Mops and Hepes buffers is characterized by a lag phase that becomes shorter with increasing FeCl2 concentration and pH. During iron oxidation in these buffers a yellow colour develops in the solution. When the reaction is conducted in the presence of nitro blue tetrazolium (NBT), blue formazan is formed. Of the many OH scavengers tested, mannitol and sorbitol are most effective in inhibiting Fe2+ oxidation, yellow colour development and NBT reduction. Some inhibition was also noted with catalase. The iron product of the oxidative reaction differs from Fe3+ in its absorption spectrum and its low reactivity with thiocyanate. Similar results are obtained when iron autoxidation is studied in unbuffered solutions brought to alkaline pH with NaOH. In phosphate buffer, no lag phase is evident and the absorption spectrum of the final solution is identical to that of Fe3+ in this buffer. The iron product reacts immediately with thiocyanate. When iron oxidation is conducted in the presence of NBT the formation of formazan is almost undetectable. Of the many compounds tested only catalase inhibits iron autoxidation in this buffer. The sequence of reactions leading to iron autoxidation in Good-type buffers thus resembles that occurring in unbuffered solutions brought to alkaline pH with NaOH and greatly differs from that occurring in phosphate buffer. These results are in agreement with the observation that these buffers have very low affinity for iron. The data presented define experimental conditions where Fe2+ is substantially stable for a considerable length of time in Mops buffer.

Evasion of macrophage microbicidal mechanisms by mature sporozoites ofPlasmodium yoelii yoelii

Sporozoites of Plasmodium yoelii yoelii were incubated for 40 min with BALB/c peritoneal macrophages in the presence of nitro-blue tetrazolium (NBT). While immature oocyst sporozoites triggered the macrophage respiratory burst, as visualized microscopically by the localized reduction of NBT to insoluble formazan, 97.6% of mature salivary gland sporozoites did not induce such a response. The macrophage oxidative response was also induced by 82.7% of heat-inactivated and 95.7% of trypsin-treated salivary gland sporozoites. The relationship of these results to the infectivity and immunogenicity of malarial sporozoites is discussed.

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase.

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

Artifactual staining of proteins on polyacrylamide gels by nitrobluetetrazolium chloride and phenazine methosulfate

Different purified proteins were shown to give purple formazan bands corresponding to the protein stain following electrophoresis on polyacrylamide gels, in the presence of nitrobluetetrazolium (NBT) and phenazine methosulfate (PMS). Both PMS and NBT are needed for formazan production which has a favorable pH at 8.5. Sulfhydryl blockers in the incubation medium inhibited this color development to different extents. While proteins with free SH groups like bovine serum albumin, ovalbumin, and urease showed this pyridine nucleotide independent artifact, nonthiol proteins, viz., bovine pancreatic ribonuclease A, and riboflavin-binding protein from chicken egg white failed to do so. The nonenzymatic formazan formation observed with different proteins could also be shown in an in vitro assay system. It is clear that the “nothing dehydrogenase” phenomenon observed in several cases may be due to the thiol group-mediated artifactual staining of proteins.

ENZYMATIC OXIDATION OF DIETHYLDITHIOCARBAMATE BY XANTHINE OXIDASE AND ITS COLORIMETRIC ASSAY*

Diethyldithiocarbamate is oxidized in vitro by purified cream xanthine oxidase in the presence of nitro blue tetrazolium and phenazine methosulfate. Disulfiram is formed during the enzymatic or nonenzymatic oxidation. When diethyldithiocarbamate is mixed nonenzymatically with nitro blue tetrazolium, the dye is rapidly reduced, but only in the presence of certain organic solvents. This reaction can be used as a convenient colorimetric assay for diethyldithiocarbamate, which gives a stable color over a wide concentration range.